Methods in molecular biology vol 1575 synthetic antibodies methods and protocols

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Methods in Molecular Biology 1575 Thomas Tiller Editor Synthetic Antibodies Methods and Protocols Methods in Molecular Biology Series Editor John M Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Synthetic Antibodies Methods and Protocols Edited by Thomas Tiller MorphoSys AG, Discovery Alliances & Technologies, Planegg, Germany Editor Thomas Tiller MorphoSys AG, Discovery Alliances & Technologies Planegg, Germany ISSN 1064-3745     ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6855-8    ISBN 978-1-4939-6857-2 (eBook) DOI 10.1007/978-1-4939-6857-2 Library of Congress Control Number: 2017933077 © Springer Science+Business Media LLC 2017 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A Dedication Dedicated to Isi, Josh & Family .and Rea Thanks for inspirational, wise, and humorous support Preface Antibodies are important tools that are used extensively in basic biomedical research, in diagnostics, and in the treatment of diseases Traditionally, the production of antibodies relies on the immunization of an animal For example, for the generation of monoclonal antibodies by the hybridoma technology, usually mice and rats are preferred For polyclonal antibody production, larger mammals (e.g., rabbits, sheep, and goats) are used as the relatively huge amount of serum that can be collected from these animals serves as a rich source for antibody purification These antibodies are all based on an immunoglobulin scaffold and are derived from a genuine in vivo immune response Despite their widespread applications as detection, diagnostic, and therapeutic agents, in vivo-generated polyclonal and monoclonal antibodies bear some limitations For example, polyclonal antibodies as detection reagents are not only prone to batch-to-batch variability but also contain significant amounts of nonspecific antibodies Furthermore, due to their inadequate characterization, it is not surprising that many experimental results that are obtained with polyclonal antibodies are often not reproducible In contrast, hybridoma-derived monoclonal antibodies are considered to be perfectly defined reagents with unique specificities Very often, however, they secrete additional light and/or heavy chains, which makes it cumbersome to evaluate if the binding behavior of the hybridoma-­derived mAb is intrinsic to the mAb from the target B cell or due to artificial chain combinations caused by the presence of the additional chains derived from the fusion cell line Furthermore, hybridoma cells can lose expression, are prone to mutations, and thus require frequent retesting The restrictions of these traditional in vivo-generated antibodies have been overcome by modern synthetic recombinant in vitro antibody technologies One of the most significant difference between naturally occurring and synthetic immunoglobulins per se is the way these two groups are generated Naturally occurring immunoglobulins are generated in vivo by processes of V(D)J recombination and somatic hypermutation of the B cell antigen receptor during B cell development and differentiation and its secretion as soluble immunoglobulin by plasma cells Synthetic antibodies on the other hand can be defined in general as affinity reagents engineered entirely in vitro, thus completely eliminating animals from the production process (Although this definition might get blurred, e.g., by processes such as antibody humanization, which basically is the replacement of frameworks of a murine antibody generated in vivo with their human counterparts by recombinant genetic engineering in vitro Therefore, a humanized antibody could be considered as “semisynthetic”) Synthetic affinity reagents include recombinantly produced immunoglobulin antibodies derived from combinatorial antibody libraries (i.e., antibody libraries built on in silico-­ designed and chemically defined diversity on the basis of synthetic oligonucleotides) and so-called antibody mimetics that are based on alternative protein/polypeptide scaffolds In addition, the term “synthetic antibody” is also often used to describe affinity reagents that are different from protein/polypeptides but share typical antibody characteristics such as diversity and specific binding affinities For example, aptamers as a class of small nucleic vii viii Preface acid ligands are composed of RNA or single-stranded DNA oligonucleotides Like antibodies, aptamers interact with their corresponding targets with high specificity and affinity An example of synthetic “plastic antibodies” are molecularly imprinted polymers (MIPs), which are polymeric matrices obtained by a technique called molecular imprinting technology to design artificial receptors with a predetermined selectivity and specificity for a given analyte MIPs are able to mimic natural recognition entities, such as antibodies and biological receptors This volume on Synthetic Antibodies aims to present a set of protocols useful for research in the field of recombinant immunoglobulin and alternative scaffold engineering, aptamer development, and generation of MIPs Part I includes methods that deal with amino acid-based synthetic antibodies Brief protocols about the generation of antibody libraries are detailed, as well as techniques for antibody selection, characterization, and validation This section is completed by a brief description of a bioinformatics platform that supports antibody engineering during Research and Development Part II contains basic procedures about the selection and characterization of aptamer molecules, and Part III describes fundamental processes of MIP generation and application I would like to express my sincere thanks to all contributing authors for sharing their research expertise Without their support, this volume would not have been possible Many thanks to John M. Walker for the invitation to edit this volume on “Synthetic Antibodies” and to Monica Suchy and Patrick Marton from Springer for helpful advice and for publishing this book Planegg, Germany Thomas Tiller Contents Preface vii Contributors xi Part I Amino Acid-Based Synthetic Antibodies   Antibody Mimetics, Peptides, and Peptidomimetics Xiaoying Zhang and Thirumalai Diraviyam   Construction of a scFv Library with Synthetic, Non-­combinatorial CDR Diversity Xuelian Bai and Hyunbo Shim   Enzymatic Assembly for scFv Library Construction Mieko Kato and Yoshiro Hanyu   Directed Evolution of Protein Thermal Stability Using Yeast Surface Display Michael W Traxlmayr and Eric V Shusta   Whole Cell Panning with Phage Display Yvonne Stark, Sophie Venet, and Annika Schmid   Generating Conformation and Complex-Specific Synthetic Antibodies Marcin Paduch and Anthony A Kossiakoff   High-Throughput IgG Conversion of Phage Displayed Fab Antibody Fragments by AmplYFast Andrea Sterner and Carolin Zehetmeier   Utilization of Selenocysteine for Site-Specific Antibody Conjugation Xiuling Li and Christoph Rader   Solubility Characterization and Imaging of Intrabodies Using GFP-Fusions Emilie Rebaud, Pierre Martineau, and Laurence Guglielmi 10 Antibody Validation by Immunoprecipitation Followed by Mass Spectrometry Analysis Helena Persson, Charlotta Preger, Edyta Marcon, Johan Lengqvist, and Susanne Gräslund 11 Novel HPLC-Based Screening Method to Assess Developability of Antibody-Like Molecules Neeraj Kohli and Melissa L Geddie 12 Glycosylation Profiling of α/β T Cell Receptor Constant Domains Expressed in Mammalian Cells Kai Zhang, Stephen J Demarest, Xiufeng Wu, and Jonathan R Fitchett 13 A Proximity-Based Assay for Identification of Ligand and Membrane Protein Interaction in Living Cells Hongkai Zhang and Richard A Lerner ix 15 31 45 67 93 121 145 165 175 189 197 215 x Contents 14 A Biotin Ligase-Based Assay for the Quantification of the Cytosolic Delivery of Therapeutic Proteins 223 Wouter P.R Verdurmen, Marigona Mazlami, and Andreas Plückthun 15 Data-Driven Antibody Engineering Using Genedata Biologics™ 237 Maria Wendt and Guido Cappuccilli Part II Nucleotide-Based Synthetic Antibodies: Aptamers 16 Selection of Aptamers Against Whole Living Cells: From Cell-SELEX to Identification of Biomarkers Nam Nguyen Quang, Anna Miodek, Agnes Cibiel, and Frédéric Ducongé 17 Rapid Selection of RNA Aptamers that Activate Fluorescence of Small Molecules Grigory S Filonov 18 An Enzyme-Linked Aptamer Sorbent Assay to Evaluate Aptamer Binding Matthew D Moore, Blanca I Escudero-Abarca, and Lee-Ann Jaykus 19 Incorporating Aptamers in the Multiple Analyte Profiling Assays (xMAP): Detection of C-Reactive Protein Elyse D Bernard, Kathy C Nguyen, Maria C DeRosa, Azam F Tayabali, and Rocio Aranda-Rodriguez 253 273 291 303 Part III Moleculary Imprinted Polymers 20 Transferring the Selectivity of a Natural Antibody into a Molecularly Imprinted Polymer Romana Schirhagl 21 Preparation of Molecularly Imprinted Microspheres by Precipitation Polymerization Tibor Renkecz and Viola Horvath 22 Generation of Janus Molecularly Imprinted Polymer Particles Xiantao Shen, Chuixiu Huang, and Lei Ye 23 Surface Engineering of Nanoparticles to Create Synthetic Antibodies Linda Chio, Darwin Yang, and Markita Landry 24 H5N1 Virus Plastic Antibody Based on Molecularly Imprinted Polymers Chak Sangma, Peter A Lieberzeit, and Wannisa Sukjee 25 Replacement of Antibodies in Pseudo-ELISAs: Molecularly Imprinted Nanoparticles for Vancomycin Detection Francesco Canfarotta, Katarzyna Smolinska-Kempisty, and Sergey Piletsky 26 Cell and Tissue Imaging with Molecularly Imprinted Polymers Maria Panagiotopoulou, Stephanie Kunath, Karsten Haupt, and Bernadette Tse Sum Bui 325 341 353 363 381 389 399 Index 417 Contributors Rocio Aranda-Rodriguez  •  Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON, Canada Xuelian Bai  •  Department of Life Science, Ewha Womans University, Seoul, Korea Elyse D. Bernard  •  Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON, Canada Bernadette Tse Sum Bui  •  CNRS Enzyme and Cell Engineering Laboratory, Sorbonne Universités, Université de Technologie de Compiègne, Compiègne Cedex, France Francesco Canfarotta  •  MIP Diagnostics Ltd., University of Leicester, Leicester, UK Guido Cappuccilli  •  Genedata AG, Basel, Switzerland Linda Chio  •  Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA Agnes Cibiel  •  Commissariat l’Energie Atomique et aux Energies Alternatives (CEA), Département de la Recherche Fondamentale (DRF), Institut d’Imagerie Biomédicale (I2BM), Molecular Imaging Center (MIRCen), CNRS UMR 9199, Neurodegenerative Diseases Laboratory (LMN), Université Paris-Sud, Université Paris-Saclay, Fontenay-aux-Roses, France Stephen J. Demarest  •  Eli Lilly Biotechnology Center, San Diego, CA, USA Maria C. DeRosa  •  Chemistry Department, Carleton University, Ottawa, ON, Canada Thirumalai Diraviyam  •  College of Veterinary Medicine, Northwest Agriculture and Forestry University, Yangling, Shaanxi, China; Department of Microbiology, Karpagam University, Coimbatore, Tamil Nadu, India Frédéric Ducongé  •  Commissariat l’Energie Atomique et aux Energies Alternatives (CEA), Département de la Recherche Fondamentale (DRF), Institut d’Imagerie Biomédicale (I2BM), Molecular Imaging Center (MIRCen), CNRS UMR 9199, Neurodegenerative Diseases Laboratory (LMN), Université Paris-Sud, Université ParisSaclay, Fontenay-aux-Roses, France Blanca I. Escudero-Abarca  •  Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University, Raleigh, NC, USA Grigory S. Filonov  •  Essen Bioscience, Ann Arbor, MI, USA Jonathan R. Fitchett  •  Eli Lilly Biotechnology Center, San Diego, CA, USA Melissa L. Geddie  •  Merrimack Pharmaceuticals, Inc., Cambridge, MA, USA Susanne Gräslund  •  Structural Genomics Consortium, Department of Biochemistry and Biophysics, Karolinska Institutet, Solna, Sweden Laurence Guglielmi  •  IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France; INSERM, U1194, Montpellier, France; Université de Montpellier, Montpellier, France; Institut régional du Cancer de Montpellier, Montpellier, France Yoshiro Hanyu  •  Structure Physiology Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan Karsten Haupt  •  CNRS Enzyme and Cell Engineering Laboratory, Sorbonne Universités, Université de Technologie de Compiègne, Compiègne Cedex, France xi MIP Imaging 3.1.2  Synthesis of MIPGlcA 405 Weigh 4.27 mg (0.022 mmol) of the template GlcA and 5.46 mg (0.022 mmol) of AB in a mL glass vial Add mL of anhydrous DMSO and incubate the mixture for h on a tube-­rotator to form the complex GlcA-AB (see Note 4) Mix together 5.6 mg (0.066 mmol) of the co-monomer MAM, 83.1 μL (0.44 mmol) of the cross-linker EGDMA, 0.0055 mmol ABDV (pipet 0.524 mL from a stock solution of 3.4 mg ABDV in 1.3 mL DMSO), 1.3 μmol of methacryloxyethyl thiocarbonyl rhodamine B (pipet 0.27 mL from a stock solution of 3 mg in mL DMSO) (see Note 5) and add to the AB-GlcA complex Synthesize a control non-imprinted polymer (NIP) in the same way but without the addition of the template (see Notes and 7) Sonicate the prepolymerization mixture in an ultrasonic bath for 15 min and purge for 2 min with nitrogen Seal the vials with a septum and Parafilm, place them in a water bath set at 48 °C and leave the polymerization to proceed for 18 h In order to remove the template from the polymeric matrix, wash the MIP three times with 1 M HCl, three times with methanol–acetic acid (1:9, v/v) and three times with methanol Finally, dry the particles overnight under vacuum (see Note 8) 3.2  Synthesis of  MIP-­Functionalized QDs for Multiplexed Cell Imaging 3.2.1  Synthesis of a Hydrophilic CrossLinked First Shell Synthesize green InP/ZnS QDs as described [34]: Add indium chloride (0.1 mmol), stearic acid (0.1 mmol), hexadecylamine (0.2 mmol) and zinc undecylenate (0.1 mmol) to 1-octadecene (2 mL) Deoxygenate the mixture repeatedly and refill with nitrogen gas to provide a water-free and oxygen-free reaction atmosphere, then heat to 270 °C with stirring On reaching 270 °C, inject rapidly mL of 0.1 M tris(trimethylsilyl) phosphine in 1-octadecene Hold the mixture at 240 °C for 20 min, then cool to room temperature Open the flask and add zinc diethyldithiocarbamate (0.2 mmol) and zinc undecylenate (0.2 mmol) Re-deoxygenate the mixture and place it under nitrogen gas, then heat to 180 °C for 10 min and to 240 °C for 20 min Cool the reaction to room temperature, then add mL toluene and centrifuge the solution at 2200 × g for 5 min Pour off the clear QD solution and add ethanol until the QDs precipitate Centrifuge the mixture at 2200 × g for 15 min Remove the supernatant and resuspend the QDs in toluene Repeat the precipitation with ethanol for three times to ensure the removal of synthetic residues Finally, resuspend the QDs in toluene and store them at °C in the dark until use In a mL glass vial containing 16.4 mg (0.097 mmol) of EbAM, 26.5 μL (0.22 mmol) of HEMA and 100 μg green-­QDs 406 Maria Panagiotopoulou et al (100 μL from the mg/mL QD solution) (see Note 9) add 300 μL DMSO–toluene (1:1, v/v), 20 μL of eosin Y (10 mM in DMSO–toluene (1:1, v/v)), and 10 μL of TEA (72 mM in DMSO–toluene (1:1, v/v)) For the red QDs, follow the same procedure, but use 20 μL methylene blue (10 mM DMSO– toluene (1:1, v/v)) as the initiator dye Seal the vial with an airtight septum and sonicate in an ultrasonic bath for 15 min Purge the mixture with nitrogen for 2 min and use a UV lamp placed at ~2 cm from the vial in order to initiate the polymerization by irradiation at 365 nm Leave the samples to polymerize for h After polymerization, transfer the content of the vial to mL polypropylene microcentrifuge tubes Subsequently, add 500 μL of DMSO–toluene (1:1, v/v) and ultrasonicate the HEMA-­ QDs particles, then sediment them by centrifugation for 15 min at 17,500 × g Wash the particles four times with 800 μL DMSO–toluene (1:1, v/v) and twice with water Photobleach the eosin Y (or methylene blue) trapped inside the particles overnight with a fluorescent tube and, finally, dry the nanoparticles under vacuum 3.2.2  Synthesis of a Second Shell (MIPGlcA-QDs or MIPNANA-QDs) Incubate 5.46 mg AB (0.022 mmol) and 4.27 mg (0.022 mmol) GlcA (or 6.8 mg (0.022 mmol) NANA) for h in mL DMSO on a tube-rotator (see Notes and 10) Following the pre-incubation step, transfer the contents of the vial to the mL glass vial containing the HEMA-QDs Subsequently, add 80 μL (0.423 mmol) of EGDMA, 5.62 mg (0.066 mmol) MAM, 20 μL of eosin Y (10 mM) and 10 μL of TEA (72 mM) In the case of the HEMA-red QDs follow the same procedure, but use 20 μL methylene blue (10 mM in DMSO–toluene (1:1, v/v)) as the initiator dye Synthesize a control non-imprinted polymer in the same way but without the addition of the template Seal the vial with an airtight septum and sonicate in an ultrasonic bath for 15 min Purge the mixture with nitrogen for 2 min and use a UV lamp placed at ~2 cm from the vial in order to initiate the polymerization by irradiation at 365 nm Leave the samples to polymerize for h After polymerization, transfer the content to mL polypropylene microcentrifuge tubes and wash the particles three times with methanol–acetic acid (9:1, v/v) followed by three times with 100 mM NH3 in water–methanol (7:3, v/v), twice with water and three times with methanol Photobleach the eosin Y (or methylene blue) trapped inside the particles overnight with a fluorescent tube and, finally, dry the nanoparticles overnight under vacuum (see Notes 11 and 12) MIP Imaging 3.3  Cell Imaging with MIPs 407 Prepare cell samples for imaging: Human adult low Calcium high Temperature (HaCaT) cells are cultured in Dulbecco’s Modified Eagle Medium high glucose with 10% FBS and 1% penicillin/streptomycin at 37 °C, 5% CO2, and 100% humidity in 75 cm2 sterile cell culture flasks Passage the cells when confluent For that, add 16 mL of a solution of PBS containing 0.05% EDTA to the flask with the cells and incubate them together for 10 min at 37 °C. Aspirate the PBS solution containing EDTA. Add mL of 0.25% trypsin–EDTA and incubate for 4 min at 37 °C (see Note 13) In order to stop the trypsinization, add mL of culture medium and transfer the obtained cell suspension to a 15 mL conical sterile polypropylene centrifuge tube Centrifuge for 5 min at 400 × g Resuspend the cell pellet in mL of culture medium and transfer 2.5 mL of the cell suspension to a new 75 cm2 sterile cell culture flask Add 17.5 mL medium to the new cell culture flask to get a final volume of 20 mL For the microscopic studies, the cells are cultured in 12-well plates (well diameter 22.1 mm) equipped with round glass coverslips (diameter 12 mm) (see Note 14) Pipette 100 μL of × 105 suspended HaCaT cells (see Note 15) onto each coverslip After h of incubation, add mL of culture medium to the cells Afterwards, let them grow to confluency for 48–60 h Wash each coverslip with confluent HaCaT cells in the 12-well plates three times with mL PBS and fix the cells at room temperature for 10 min with 600 μL paraformaldehyde 3% (w/v) in PBS (see Note 16) To stop fixation and reduce nonspecific interactions, incubate each cell sample three times with mL 20 mM glycine in PBS for 20 min at room temperature (see Note 17) and finally wash them three times with mL PBS Wash the cells three times with mL methanol–water (1:30, v/v) and then incubate them with mL of tip-sonicated polymer suspensions: 27 μg/mL rhodamine-labeled MIPGlcA (Fig. 2a) or 60 μg/mL MIPGlcA-QDs (green QDs) (Fig. 2b) or 60 μg/mL MIPNANA-QDs (red QDs) (Fig. 2c) at 37 °C for 90 min For multiplexed cell imaging, add simultaneously mL of 60 μg/mL MIPGlcA-QDs and mL of 60 μg/mL MIPNANA-­QDs to each well and then let them incubate at 37 °C for 90 min (see Note 18) (Fig. 2d) Afterwards, wash each fixed cell layer three times with mL methanol–water (1:30, v/v) (see Note 19) When quantitative analysis is not required, perform the additional staining of the cell membrane with the DiO dye, after prior MIP incubation and washing of the cells For that, mix 200 μL FBS with 100 μL DiO (100 μM stock solution in 408 Maria Panagiotopoulou et al Fig Confocal images on fixated keratinocytes for the localization of (a) rhodamine-labeled MIPGlcA (red); (b) MIPGlcA-QDs (green, in some cells MIPGlcA-QDs were observed in nuclear clefts; (c) MIPNANA-QDs (red), the particles are mainly localized extracellularly and pericellularly (d) Simultaneous multiplexed labeling of both GlcA and NANA by MIPGlcA-QDs (green) and MIPNANA-QDs (red) Nuclear staining is performed with DAPI (blue) and membrane staining in the case of (a) is performed with Dio (green) Scale bars: 25 μm Images are reproduced with permissions from refs 18, 19, 26 DMSO) Afterwards, add 800 μL water and use 300 μL of this solution to stain the fixed, washed cells by incubating for 30 min at room temperature (see Note 20) After staining, wash the samples three times with mL water When quantitative analysis is required, not perform cell membrane or nuclear staining As a final step, mount the samples for fluorescence microscopy imaging on a microscope slide with μL mounting medium (see Note 21) For the staining of the cell nucleus, mix in a ratio 1:10 a stock solution of mg/mL DAPI in water with mounting medium Place μL from this solution on a microscope slide to mount the cells on the coverslips (see Note 22) After min, image capturing takes place MIP Imaging 3.4  Tissue imaging with MIPs 409 Adult skin specimens are collected by autopsy Slice the samples in μm thick sections in a cryostat microtome, transfer them to adhesive microscope slides and dry them for 30 s in an oven maintained at 70 °C to ensure good sample attachment to the glass surface Subsequently, fix the tissue sections for 10 min with cold acetone (−10 °C) and wash them three times with PBS Prior to MIPGlcA incubation, wash the samples three times with mL methanol–water (1:30, v/v) and then incubate them with mL of a tip-sonicated polymer suspension of 27 μg/mL polymer in methanol–water (1:30, v/v) at 37 °C for 90 min Afterwards, wash each tissue sample three times with mL methanol–water (1:30, v/v) and mount for fluorescence microscopy in 20 μL mounting medium (Fig. 3) In the cases where quantitative analysis is not required, perform additional staining of the cell nucleus with 20 μL mounting medium containing 100 μg/mL DAPI (see Note 23) 3.5  Acquisition and Analysis of Images 3.5.1  Epifluorescence Microscopy Imaging Quantitative cell imaging using the rhodamine-labeled MIPGlcA, MIPGlcA-QDs, or MIPNANA-QDs: Epifluorescence images are captured with a Leica DMI 6000B microscope, filter set A4, L5, TX2, N PLAN L 20.0 × 0.40 DRY, HCX PL FLUOTAR 40.0 × 0.60 DRY, HCX FLUOTAR 63.0 × 0.70 DRY, and HCX FLUOTAR 100.0 × 1.30 OIL objectives with 20×, 40×, 63×, and 100× magnification Fig Confocal image for the localization of rhodamine-labeled MIPGlcA (red) in human skin specimens Nuclear staining is performed with DAPI (blue) The MIPGlcA particles bound to the skin tissue are mainly localized in the basal layer of the epidermis and the papillary dermis Scale bar: 100 μm 410 Maria Panagiotopoulou et al Images are captured using exactly the same settings concerning light intensity and exposure time in 16-Bit Tiff format Only confluent cell layers are examined for quantification studies and when quantitative analysis is carried out, nuclear staining is not performed From each sample, at least four images are captured with the Leica Application Suite (LAS) software and each cell sample is at least prepared in quadruplicate All fluorescence intensities are determined with ImageJ. For image analysis, if needed perform prior background subtraction in order to determine the fluorescence signal coming only from the particles As a control for the background signal, samples with fixed cells are used and the average background signal at four different areas of each gray value image is subtracted prior to quantification Furthermore, a slight difference in the fluorescence intensity from MIP and NIP particles is corrected during the quantification using ImageJ 3.5.2  Confocal Microscopy Imaging Confocal images are captured with a Zeiss LSM 710, AxioObserver A Plan-Apochromat 63×/1.40 OIL DIC M27 objective and 405, 488, and 543 nm lasers are used for excitation of DAPI, DiO, and rhodamine, respectively, for all images The samples used for confocal microscopy are stained for hyaluronan using the rhodamine-labeled MIPGlcA (red), for the cell membrane using Dio (green) and for the cell nucleus using DAPI (blue) Alternatively, when staining for hyaluronan using the MIPGlcA-QDs (green) or for sialylated sites using the MIPNANA-­QDs (red), not perform cell membrane staining Stain the cell nucleus in the latter case with DAPI (blue) (see Note 24) Capture all the confocal images from the middle Z of the samples 4  Notes Successful cell culture depends on keeping the cells free from contamination by microorganisms such as bacteria, fungi, and viruses During all cell manipulations, follow the aseptic technique in order to reduce the probability of contamination The elements of aseptic technique are a sterile work area, good personal hygiene, sterile reagents (autoclave, spraying with 70% ethanol etc.) and media, and sterile handling Sterilize the solution of PBS containing 0.05% EDTA, in an autoclave before use The yield of the synthesis of 4-acrylamidophenyl(amino) methaniminium acetate (AB) is ~80% MIP Imaging 411 The stoichiometry and the association constant of the AB-­ GlcA complex are deduced from 1H NMR titration studies in DMSO-d6, yielding a 1:1 ratio and a high association constant Ka of 7.1 × 103 M−1 The molar ratio of methacryloxyethyl thiocarbonyl rhodamine B with respect to the other monomers was optimized to maximize the fluorescence intensity of the particles (optimal ratio: 1:0.05 AB:rhodamine) Higher dye content resulted in lower brightness due to reabsorption or energy transfer The degree of cross-linking (mol of EGDMA upon total number of mol of EGDMA and functional monomers) in the described protocol is 83%, with a molar ratio of GlcA:AB:MAM:EGDMA of 1:1:3:20 and a total concentration of monomers of 5% (mass of monomers relative to total mass of solvent and monomers) The initiator, ABDV, is used in a concentration of 0.56 mol% with respect to the total number of mol of double bonds in the polymerization mixture MIPGlcA and NIPGlcA particles with diameters of approximately 400 nm (less than 10% deviation between MIP and NIP) with good monodispersity are obtained using this precipitation polymerization protocol The particle size is chosen to avoid possible internalization of the particles, so that only the extracellular hyaluronan is targeted The recognition properties of rhodamine-labeled MIPGlcA are evaluated by equilibrium radioligand binding assays using [14C]glucuronic acid (Biotrend Chemikalien GmbH, Köln, Germany) in methanol–water (9:1, v/v) and in pure water, where maximum imprinting factors (IFmax) of 2.2 and 3.2 are respectively observed (IF = binding to the MIP/binding to the NIP) Sonicate the mg/mL QD stock solution (green or red QDs) in toluene for 15 min with a microtip (Branson Sonifier 250) prior to addition in the prepolymerization mixture 10 The stoichiometry and the association constant of the AB-­ NANA complex are deduced from 1H NMR titration studies in DMSO-d6, yielding a 1:1 ratio and a high Ka of 41 × 103 M−1 11 The described protocol results in an average particle size of 125 ± 17 nm for both MIPGlcA-QDs and MIPNANA-QDs, enabling the staining of the intracellular glycosylation sites 12 The recognition properties of MIP/NIPGlcA-QDs and MIP/ NIPNANA-QDs are evaluated by equilibrium radioligand binding assays using [14C]glucuronic acid and [6-3H]sialic acid respectively (Biotrend Chemikalien GmbH, Köln, Germany) in water, where high specificity is observed 412 Maria Panagiotopoulou et al 13 In case there is any problem with the cell trypsinization, incubate the cells with the mL of 0.25% trypsin–EDTA at 37 °C for up to maximum 10 min 14 Prior to use, sterilize the coverslips by dipping in ethanol 70% and leave them to dry for 30 min under the hood 15 Cell counting is performed using a disposable hemocytometer After cell growth and trypsinization (see Subheading 3.3, steps 1–2), centrifuge the cells for 5 min at 400 × g Then resuspend the cell pellet in mL of culture medium and transfer the cell suspension to a 50 mL conical polypropylene centrifuge tube Add 15 mL culture medium in order to get a final volume of 20 mL. Before the cells have a chance to settle, withdraw 500 μL of the cell suspension into an Eppendorf tube In another Eppendorf tube, pipet 400 μL of 0.4% Trypan Blue Subsequently, transfer 100 μL of cells into the latter tube Mix gently by pipetting Afterwards, take 100 μL of the Trypan Blue-treated cells and pipet them into the well of the counting chamber of the hemocytometer, allowing the capillary forces to draw it inside Use a microscope with an objective with a 10× magnification and focus on the grid lines of the hemocytometer Count the live, unstained cells using a hand tally counter (living cells are impermeable to Trypan Blue) In order to obtain a correct count, adapt a counting system whereby cells are only counted when they are set within a square or on the right-hand or bottom boundary line Carry on counting until all sets of 16 corners are counted To obtain the number of viable cells/mL in the original cell suspension, take the average cell count from each of the sets of 16 corner squares Multiply by 104 Multiply again by to correct for the 1:5 dilution from the Trypan Blue addition 16 The fixation of cells is based on paraformaldehyde that has low background fluorescence Aldehyde fixatives react with amines and proteins to generate fluorescent products Glutaraldehyde is less suitable than formaldehyde due to generation of stronger fluorescence The simplest way to stop aldehyde-induced fluorescence is to use a fixative that does not contain an aldehyde Carnoy, Clarke, and methacarn solutions are examples, but are used only for subsequent paraffin sectioning The remedy to the aldehyde problem is aldehyde blocking 17 When fixatives react and cross-link with protein molecules, lots of free aldehyde groups remain These cell/tissue-bound free aldehyde groups will combine covalently with any amino group offered to them, including terminal and side-chain (lysine) amino groups of proteins being used as histochemical reagents, which means all antibodies, all lectins and all enzymes This way, even a highly specific monoclonal primary antibody may bind at sites that contain basic proteins but not MIP Imaging 413 the antigen of interest This is why, aldehyde blocking is necessary This is done by reducing the −CHO groups to –CH2OH with sodium borohydride or by feeding them small-molecule amines, e.g., glycine 18 Incubation of the rhodamine-labeled MIPGlcA with the cells was also carried out at room temperature with and without agitation In both cases, particle aggregation was observed rendering these samples unsuitable for cell imaging 19 The selectivity of MIPGlcA is confirmed by competitive binding assays comparing the binding of GlcA to that of other monosaccharides such as glucose, galactose, N-­acetylglucosamine, N-acetylgalactosamine and NANA, present at the terminal parts of glycoproteins or glycolipids that could potentially interfere during cell imaging; less than 1% cross-reactivity is observed In the case of the MIPNANA, selectivity is also confirmed by competitive equilibrium binding assays using [6-3H]sialic acid (Biotrend Chemikalien GmbH, Köln, Germany) which show less than 10% cross-­reactivity with GlcA and negligible crossreactivity with other terminal sugars, namely N-acetylglucosamine, glucose, galactose, and N-acetylgalactosamine These results indicate that multiplexed cell imaging using the MIPs described in this protocol is possible 20 Cell membrane staining with DiO was also carried out for 15 min, but better staining results are achieved with 30 min incubation 21 In all cases, an IFmax ~ is observed on the cells, verifying the imprinting effect To further prove the specificity of the MIPs, the GlcA and NANA terminal moieties are cleaved off enzymatically with the use of hyaluronidase and neuraminidase, respectively After the enzymatic treatment, there is no significant difference anymore between MIPs and NIPs labeling 22 Apply transparent nail polish on the edges of the coverslips in order to ensure sample attachment to the microscope slides 23 The MIPGlcA particles bound to the skin tissue are mainly localized in the basal layer of the epidermis and the papillary dermis Lower amounts of particles can be found in the cornified and granular cell layer and even lower amounts in the spinous cell layer This is in agreement with and the control results obtained with FITC-labeled hyaluronic acid binding protein applied to tissue samples from the same batch and prepared in the same way as well as to the descriptions of hyaluronan localization in the literature [35–37] 24 Confocal images for the rhodamine-labeled MIPGlcA are corrected by subtracting the rhodamine signal from the green channel as there is spectral overlap with λex(rhodamine) = 488 nm The subtraction was performed with ImageJ 414 Maria Panagiotopoulou et al Acknowledgment The authors thank the European Regional Development Fund and the Regional Council of Picardie (co-­funding of equipment under CPER 2007–2013), the European Union (FP7 Marie Curie Actions, ITN SAMOSS, PITN-2013–607590), and the french embassy in Germany (postdoctoral scholarship of S.K.), for financial support The authors thank Jörg Sänger and the Institute of Pathology Bad Berka (Germany) for providing tissue samples and for tissue imaging References Moremen KW, Tiemeyer M, Nairn AV (2012) Vertebrate protein glycosylation: diversity, synthesis and function Nat Rev Mol Cell Biol 13(7):448–462 Spiro RG (2002) Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds Glycobiology 12(4):43R–56R Bard F, Chia J (2016) Cracking the glycome encoder: signaling, trafficking, and glycosylation Trends Cell Biol 26(5):379–388 Ohtsubo K, Marth JD (2006) Glycosylation in cellular mechanisms of health and disease Cell 126:855–867 Rudd PM, Elliott T, Cresswell P, Wilson IA, Dwek RA (2001) Glycosylation and the immune system Science 291:2370–2376 Gopaul KP, Crook MA (2006) Sialic acid: a novel marker of cardiovascular disease? Clin Biochem 39(7):667–681 Hascall VC, Majors AK, De la Motte CA, Evanko SP, Wang A, Drazba JA et al (2004) Intracellular hyaluronan: a new frontier for inflammation? Biochim Biophys Acta 1673: 3–12 Varki NM, Varki A (2007) Diversity in cell surface sialic acid presentations: implications for biology and disease Lab Invest 87:851–857 Seton-Rogers S (2012) Metastasis multitasking hyaluronic acid Nat Rev Cancer 12:228–228 10 Büll C, Stoel MA, Den Brok MH, Adema GJ (2014) Sialic acids sweeten a tumor's life Cancer Res 74:3199–3204 11 Kawamura A, Kijima-Suda I, Sugimoto M, Itoh M, Takada K, Tomita K et al (1990) A monoclonal antibody to free N-acetylneuraminic acid Biochim Biophys Acta 1033:201–206 12 De la Motte CA, Drazba JA (2011) Viewing hyaluronan: Imaging contributes to imagining new roles for this amazing matrix polymer J Histochem Cytochem 59:252–257 13 Sterner E, Flanagan N, Gildersleeve JC (2016) Perspectives on anti-glycan antibodies gleaned from development of a community resource database ACS Chem Biol 11(7):1773–1783 14 Bowen JL, Manesiotis P, Allender CJ (2013) Twenty years since ‘antibody mimics’ by moelcular imprinting were first proposed: A critical perspective Mol Imprinting 1:35–40 15 Haupt K, Linares AV, Bompart M, Tse Sum Bui B (2012) Moelcularly imprinted polymers Top Curr Chem 325:1–28 16 Alexander C, Andersson HS, Andersson LI, Ansell RJ, Kirsch N, Nicholls IA et al (2006) Molecular imprinting science and technology: a survey of the literature for the years up to and including 2003 J Mol Recognit 19:106–180 17 Takeuchi T, Sunayama H (2015) Molecularly imprinted polymers In: Kobayashi S, Müllen K (eds) Encyclopedia of polymeric nanomaterials Springer, Berlin Heidelberg, p 1291 18 Kunath S, Panagiotopoulou M, Maximilien J, Marchyk N, Sänger J, Haupt K (2015) Cell and tissue imaging with molecularly imprinted polymers as plastic antibody mimics Adv Healthc Mater 4:1322–1326 19 Panagiotopoulou M, Kunath S, Medina-­ Rangel PX, Haupt K, Tse Sum Bui B (2016) Fluorescent molecularly imprinted polymers as plastic antibodies for selective labeling and imaging of hyaluronan and sialic acid on fixed and living cells Biosens Bioelectron doi:10.1016/j.bios.2016.07.080 20 Shinde S, El-Schich Z, Malakpour A, Wan W, Dizeyi N, Mohammadi R et al (2015) Sialic acid-imprinted fluorescent core–shell particles for selective labeling of cell surface glycans J Am Chem Soc 137:13908–13912 MIP Imaging 21 Wang S, Yin D, Wang W, Shen X, Zhu JJ, Chen HY et al (2016) Targeting and imaging of cancer cells via monosaccharide-imprinted fluorescent nanoparticles Sci Rep 6:22757–22767 22 Ton XA, Tse Sum Bui B, Resmini M, Bonomi P, Dika I, Soppera O et al (2013) A versatile fiber-optic fluorescence sensor based on molecularly imprinted microstructures polymerized in  situ Angew Chem Int Ed 52(32):8317–8321 23 Liu RY, Guan GJ, Wang SH, Zhang ZP (2011) Core-shell nanostructured molecular imprinting fluorescent chemosensor for selective detection of atrazine herbicide Analyst 136:184–190 24 Yang YQ, Niu H, Zhang H (2016) Direct and highly selective drug optosensing in real, undiluted biological samples with quantum-dot-­ labeled hydrophilic molecularly imprinted polymer microparticles ACS Appl Mater Interfaces 8(24):15741–15749 25 Wei F, Xu G, Wu Y, Wang X, Yang J, Liu L et al (2016) Molecularly imprinted polymers on dual-color quantum dots for simultaneous detection of norepinephrine and epinephrine Sens Actuators B Chem 229:38–46 26 Panagiotopoulou M, Salinas Y, Beyazit S, Kunath S, Duma L, Prost E et al (2016) Molecularly imprinted polymer-coated quantum dots for multiplexed cell targeting and imaging Angew Chem Int Ed 55:8244–8248 27 Beyazit S, Ambrosini S, Marchyk N, Palo E, Kale V, Soukka T et al (2014) Versatile synthetic strategy for coating upconverting nanoparticles with polymer shells through localized photopolymerization by using the particles as internal light sources Angew Chem Int Ed 53(34):8919–8923 28 Tang Y, Gao Z, Wang S, Gao X, Gao J, Ma Y et al (2015) Upconversion particles coated with molecularly imprinted polymers as fluo- 415 rescence probe for detection of clenbuterol Biosens Bioelectron 71:44–50 29 Xu S, Lu X (2016) Mesoporous structured MIPs@CDs fluorescence sensor for highly sensitive detection of TNT. Biosens Bioelectron 85:950–956 30 Bossi AM, Sharma PS, Montana L, Zoccatelli G, Laub O, Levi R (2012) Fingerprint-­ imprinted polymer: rational selection of peptide epitope templates for the determination of proteins by molecularly imprinted polymers Anal Chem 84(9):4036–4041 31 Rachkov A, Minoura N (2001) Towards molecularly imprinted polymers selective to peptides and proteins The epitope approach Biochim Biophys Acta 1544(1–2):255–266 32 Tammi R, Rilla K, Pienimäki J-P, MacCallum DK, Hogg M, Luukkonen M et al (2001) Hyaluronan enters keratinocytes by a novel endocytic route for catabolism J Biol Chem 276:35111–35122 33 Nestora S, Merlier F, Beyazit S, Prost E, Duma L, Baril B et al (2016) Plastic antibodies for cosmetics: molecularly imprinted polymers scavenge precursors of malodors Angew Chem Int Ed 55(21):6252–6256 34 Xu S, Ziegler J, Nann T (2008) Rapid synthesis of highly luminescent InP and InP/ZnS nanocrystals J Mater Chem 18:2653–2656 35 Tammi R, Ripellino JA, Margolis RU, Tammi M (1988) Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe J Investig Dermatol 90:412–414 36 Wang C, Tammi M, Tammi R (1992) Distribution of hyaluronan and its CD44 receptor in the epithelia of human skin appendages Histochemistry 98:105–112 37 Papakonstantinou E, Roth M, Karakiulakis G (2012) Hyaluronic acid: a key molecule in skin aging Dermatoendocrinol 4(3):253–258 Index A a-agglutinin������������������������������������������������������������������������45 Adalimumab (Humira)���������������������������������������������������������8 Adnectins�������������������������������������������������������������������������7, Ado-trastuzumab emtansine (Kadcyla)����������������������������145 Affibodies����������������������������������������������������������������� 4, 5, 7, Affinity������������������������������9–11, 31, 32, 45, 63, 98, 100, 101, 107, 108, 110–116, 165, 176, 177, 185, 189, 190, 225, 234, 245–249, 261, 267, 273–275, 277, 285, 286, 291, 292, 304, 330, 337, 368, 369, 377, 386, 391, 394 Affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS)�����������������������������������������������������������112 Affinity chromatography���������������������10, 113, 149, 157, 210 Aggregation hotspots����������������������������������������������������������������������241 Albumin binding domain�����������������������������������������������������5 Alpha/beta T cell receptor����������������������������������������197–211 Antibody developability���������������������������������������������� 189–193, 195 evaluation����������������������������������������������������������� 177, 185 fragment��������������������������������� 3, 67, 68, 72–78, 121–142, 165, 176, 177, 180, 185 library��������������������������������������� 15, 16, 27, 32, 36–37, 41, 43, 68, 103, 121, 239, 366 mimetics�������������������������������������������������������������������3–11 validation���������������������������������������94, 175–183, 185, 186 Antibody-drug conjugates (ADCs)�������������������������� 145, 146 Anticalins��������������������������������������������������������������� 4, 6, 7, 10 Asialoglycoprotein receptor (ASGPR)��������������������� 198, 209 Avimers������������������������������������������������������������������������ 4, 7, Avi-tag���������������100, 101, 115, 224, 226, 228–231, 234, 235 B Binding kinetics�������������������������������������������������������� 111, 304 Bioinformatics������������������������������������������������������������������116 Biomarkers�������������� 5, 10, 253–263, 266–268, 270, 271, 305 Biophysical properties��������������������������������������������������������16 Biotin����������������������������������� 94, 95, 100, 101, 103–106, 108, 112, 113, 115, 125, 147, 224, 225, 230, 270, 292, 293, 300, 305, 308, 317, 318, 375 Biotin ligase-based assay������������������� 223, 225–229, 231–235 Biotinylated primers����������������������������������������� 123, 140, 142 Biotinylation����������100–102, 112, 113, 224, 225, 231, 234, 235 BirA����������������������������� 94, 100, 101, 113, 224, 230, 232, 234 Bispecific antibody format������������������������������������������������250 Brentuximab vedotin (Adcetris)���������������������������������������145 Broccoli����������������������������� 274, 275, 277, 278, 280, 283, 287 C Calcein������������������������������������������������������������������� 72, 83, 89 Calnexin���������������������������������������������������������������������������197 Calreticulin�����������������������������������������������������������������������197 Cell culture������������������������������� 69, 73, 83, 113, 114, 157, 173, 177, 179–180, 216–217, 226, 337, 382, 401, 407, 410 ELISA��������������������������������������������������������������������������85 imaging������������������������ 273, 274, 400, 401, 403–409, 413 SELEX��������������������������������253–263, 266–268, 270, 271 Chromatin immunoprecipitation (ChIP)������������������� 94, 177 Colloidal stability��������������������������������������������� 190, 193, 342 Colloidosomes���������������������������������������������������������� 358, 359 Combinatorial beneficial mutations (CBM)�����������246, 248, 249 Combinatorial library������������������������������������������� 6, 246, 248 Competition selection������������������������������������������ 97, 98, 115 Complementarity determining regions (CDRs)����������������������������������������������� 16, 32, 68, 245 Complex-specific antibodies���������������������������������������93–117 Conformation-specific antibodies������������������������� 46–48, 51, 56–60, 62, 63, 93–117, 190 C-reactive protein (CRP)������������������������� 303–314, 316–321 Cross-interaction chromatography (CIC)���������������� 112, 190 Cytomegalovirus (CMV)����������������������������������������� 122, 127 Cytotoxic T lymphocyte associated antigen-4 (CTLA-4)������������������������������������������������������������6, D Database�������������������� 109, 183, 238–239, 241, 243, 247, 250 Data-dependent acquisition (DDA)������������������������� 203, 204 Data-driven antibody engineering������������237–239, 241, 243, 245–247, 249 Data-independent acquisition (DIA)�����������������������202–204 Deglycosylation���������������������������������������� 199, 204–206, 210 Degradation������������������������������������ 5, 87, 100, 141, 166, 167, 190, 227, 234, 278, 304 Designed ankyrin repeat protein (DARPins)������������� 4, 9, 10 Diagnostics�������������������� 10, 11, 121, 175, 256, 303, 382, 389 Differential scanning calorimetry (DSC)������������������� 58, 111 Differential scanning fluorimetry (DSF)������������������ 111, 190 Thomas Tiller (ed.), Synthetic Antibodies: Methods and Protocols, Methods in Molecular Biology, vol 1575, DOI 10.1007/978-1-4939-6857-2, © Springer Science+Business Media LLC 2017 417 Synthetic Antibodies: Methods and Protocols 418  Index    Differential static light scattering (DSLS)�����������������������112 Directed evolution�������������������������4, 45–46, 48–63, 285, 286 Diversity�����������������������������������5, 15–28, 32, 33, 39, 40, 42, 55, 58, 61, 68, 72, 87, 93, 98, 102, 103, 107, 116, 121, 249, 278, 281, 284 DNA aptamer�������������������������������������������������������������������314 DNA transformation������������������������������������ 58, 81, 127, 129 Drug-to-antibody ratio (DAR)���������������� 145, 153, 159–160 Duocalin�������������������������������������������������������������������������������6 E Electrospray ionization high resolution mass spectrometry (ESI-HRMS)��������������������������������������� 147, 160, 163 Enzyme-linked aptamer sorbent assay (ELASA)���������� 9, 10, 94, 107–109, 116, 221, 239, 243, 247, 253, 291, 292, 294–301, 389, 391–397 Epitope binning����������������������������������������������������������������111 Epitopes��������������������������������� 7, 10, 45, 47, 94, 99, 111, 116, 175, 305, 318, 335–338, 400 Epstein-Barr virus (EBV)������������������������������������ 9, 151, 155 Error-prone PCR�����������������������������������������������������������������5 ESI tandem mass spectrometer����������������������������������������202 Ethanol precipitation�������������������������52–54, 58, 61, 263, 279 F Fc domain�����������������������������������������������������������������������������9 Firefly luciferase������������������������������������������������������� 218, 219 Flow cytometry�������������������������������������������� 83–85, 253, 254 Fluorescence-activated cell sorting (FACS)���������� 48, 50–51, 55–58, 61, 62, 72, 83, 84, 89, 166, 167, 171, 243, 274, 277, 281, 282, 287 Fluorescent barcoding��������������������������������������������������82, 83 Fynomers���������������������������������������������������������������������� 4, 6, G Galactose���������������������������������������������������� 49, 197, 209, 413 Genedata Biologics™��������� 237–239, 241, 243, 245–247, 249 Geneticin (G418)�������������������������������������������������������������221 Glycan compositions�������������������������������� 199, 207, 208, 211 Glycan heterogeneity��������������������������������������������������������211 Glycomics�������������������������������������������������������������������������198 Glycopeptides������������������������������������198, 203, 204, 206–211 Glycoprofiling���������������������������������������������������������� 207, 211 Glycosylation��������������������������� 3, 72, 197–211, 385, 399, 400 G-protein coupled receptors (GPCRs)������������������������68, 90 Green fluorescent protein (GFP)�������������������������� 46, 49, 55, 165–168, 171, 173, 274 H H5N1 influenza virus�����������������������������������������������381–387 HEK293����������������������������������������������86, 179, 180, 185, 199 Helper phage���������������������������������� 20, 24, 35, 40, 70, 72, 75, 76, 86, 87, 95, 104, 106, 108, 114 High performance liquid chromatography (HPLC)����������������������������������41, 113, 150, 153, 181, 201, 202, 259, 292, 345, 356 High-fidelity DNA polymerase������������������������������� 131, 141 High-throughput screening (HTS)������������������ 308, 309, 317 HisTrap column������������������������������������������������������� 152, 158 HPLC-based screening method����������������������� 189–193, 195 Human epidermal growth factor receptors���������������������������5 Hydrophobic interaction chromatography (HIC)������������������������������������������� 147, 150, 159, 162 I Immobilized metal ion affinity chromatography (IMAC)����������������������������������������������������������������147 Immunofluorescence (IF)����������������������������������� 10, 177, 411 Immunogenicity����������������������������������������������� 3, 15, 68, 304 Immunoglobulin����������������������������������������� 47, 158, 159, 332 Immunoprecipitation (IP)�������������������94, 175–183, 185, 186 Imprinting������������������������ 325–330, 333, 336–338, 342, 343, 348, 354, 382, 384, 386, 389, 394, 400, 411, 413 In silico developability assessment������������������������������������240 Infrared fluorescence microscopy��������������������������������������377 Integrated assay data management���������������������������241–243 Intrabodies����������������������������������������������������������������165–173 Iodoacetamide����������������������������������147, 153, 162, 179, 181, 185, 200, 201, 203 Ion channels�����������������������������������������������������������������68, 90 J Janus particles��������������������������������������������������� 353, 354, 359 L Lentivirus�����������������������������������������������������������������217–221 Library cloning���������������������������������������������������������������� 278, 279 design��������������������������������������������������� 15, 102, 246–249 Ligation��������������� 22, 27, 42, 68, 79–81, 87, 123, 130, 133–135, 137, 138, 142, 154, 156, 160, 276, 279, 280, 288 Lipocalin������������������������������������������������������������������������������6 Lipofectamine�������������������������������������������������� 217, 219, 220 LipofectAMINE��������������������������������������������������������������171 Liquid chromatography (LC)-mass spectrometry������������������176, 179, 181–183, 186, 269 Live-cell imaging������������������������������������������������������ 273, 274 Look-through mutagenesis (LTM)������������������ 245, 246, 248 M Major histocompatibility complex (MHC)����������������������������������������������������� 46, 48, 197 Mass spectrometry (MS)��������������������������113, 160, 175–183, 185, 186, 199, 200, 202, 203, 237, 254, 329 Microscopy������������������������� 10, 112, 167, 170–171, 223, 253, 254, 329, 360, 364, 370–377, 386, 387, 401, 408–410 Microspheres������������������������������������308, 309, 312, 313, 316, 341–350, 354, 357–358 Molecularly Imprinted Polymer (MIP) particle size�����������������������������������������������������������������411 polydispersity��������������������������������������������������������������344 porosity�����������������������������������������������������������������������344 Multi-angle light scattering (MALS)�������������������������������112 Multiple-analyte profiling (xMAP)��������������������������303–321 N N-acetylgalactosamine (GalNAc)�������������������������������������413 N-acetylglucosamine (GlcNAc)������������������������������ 197, 198, 206–208, 211, 413 NanoMIPs����������������������������������������������������������������389–397 Nanoparticles�����������������������������������112, 190, 327–329, 355, 359, 363–379, 389, 391–395, 397 Next-generation sequencing (NGS)��������� 257–258, 265–267 Non-combinatorial CDR diversity�������������������������������15–28 O O-linked glycans����������������������������������������������� 198, 199, 210 O-linked glycosylation���������������������������������������������� 199, 210 Output titer�������������������������������������������������28, 74, 75, 86, 87 P Panning�����������������������������������������20, 23, 28, 32, 67–90, 121 Peptide mapping������������������������������������������������������ 201, 202, 211 Peptide N-glycosidase F (PNGase F)���������������������� 153, 160, 198, 200, 201, 203, 204, 206, 210 Phage acidic elution����������������������������������������������� 69–70, 73–74 adsorption���������������������������������������������������������������74, 85 blocking������������������������������������������������������������ 24, 69, 72 display��������������������������������������� 3, 5, 6, 28, 31, 35, 39, 40, 67–76, 78–90, 93, 98, 100, 103–107, 111, 112, 115, 121–142, 166, 169 precipitation������������������������������������������������������ 70, 75–77 production�������������������������������������������������� 70, 75–77, 87 Pharmacodynamic (PD)������������������������������������������� 145, 198 Pharmacokinetic (PK)�������������������������������������� 4, 5, 145, 198 Pickering emulsion������������������������������������������� 353–355, 358 Plasmid isolation��������������������������������47, 56–58, 61, 63, 109, 154, 155, 157, 285 Platelet-derived growth factor receptor transmembrane domain (PDGFR-TM)���������������� 215, 217, 218, 221 Polydispersity������������������������������������������������������������ 343, 344 Polyethylene glycol (PEG)���������������������������8, 20, 24, 35, 40, 41, 70, 76, 87, 95, 101, 103, 104, 106, 365 Porosity����������������������������������������������343, 344, 349, 392, 394 Post-translational modifications (PTMs)�������������� 15, 16, 99, 241, 243, 399 Precipitation polymerization������������328, 332, 341–350, 354, 402–405, 411 Synthetic Antibodies: Methods and Protocols 419 Index       Prepolymer��������������������������������325–327, 329, 330, 332, 336 Primary cell screening���������������������������������������������������71, 82 Primary multiplex cell screening����������������������������� 72, 82–85 Protein A������������������������������������������110, 117, 147, 152, 153, 157, 161, 162, 177 Protein fragment complementation (PCA)����������������������215 Protein G��������������������147, 149, 152, 153, 159, 160, 162, 177 Protein thermal stability����������������������������������� 45, 46, 48–63 Proteomics������������������������������������������������������������������������198 Proximity based assay��������������������������������������� 215–217, 220 Pseudomonas exotoxin A������������������������������������������������������5 Q Quantum dots (QDs)�����������������������������������������������400–411 Quartz crystal microbalances (QCM)������������� 327, 330–334, 384, 387 R Radioactive binding assay��������������������������������� 258, 267–268 Restriction digest����������������������������������������������� 37, 123, 130, 132–134, 136, 137, 139, 141 Restriction fragment length polymorphism (RFLP)�������257, 263–265 Retention times������������������������� 112, 150, 190, 193, 194, 206 Retrovirus�����������������������������������������������������������������167–172 Ribosome display��������������������������������������������������������������3, RNA aptamers������������������� 273–288, 305, 306, 314, 316, 317 RNA libraries����������������������������254, 256, 260–261, 275, 278 S Sandwich assay��������������������������������������������������������� 305, 318 SDS-PAGE��������������������������������������101, 102, 111, 149, 159, 227, 231–232, 234, 259, 269 Sec insertion sequence (SECIS)������������������������������ 146–148, 150–157, 161, 162 Selenocysteine (Sec)��������������������������146–158, 160, 162, 163 Selenomabs������������������������ 146, 147, 150, 156–160, 162, 163 SELEX See Systematic evolution of ligands by exponential enrichment (SELEX) Self-propelled microengine����������������������������������������������360 Simulation of CDRs Inspired by and Emulating Nature (SCIEN) principle��������������������������������������������������16 Single-walled carbon nanotubes (SWNT)�������������� 363–370, 373–379 Site-directed mutagenesis��������������������������������� 151, 155, 161 Site-specific antibody conjugation������������������������������������145 Size exclusion chromatography (SEC)��������������������� 112, 194 Small molecules������������������������������������10, 97, 111, 114, 116, 146, 215, 273–288, 326, 332 Specificity����������������������6, 10, 32, 72, 98, 107, 108, 111, 116, 165, 177, 273, 298, 299, 304, 315, 367, 391, 411, 413 Spectroscopy��������������������������������������112, 140, 190, 241, 374 Spinach������������������������������ 273, 274, 276–278, 280, 282, 283 Spot titration���������������������������������������������������������� 77–78, 87 Synthetic Antibodies: Methods and Protocols 420  Index    Stability�������������������������������9, 11, 15, 45–63, 94, 97, 98, 100, 111, 114, 146, 166, 186, 189, 190, 193, 316, 342, 364, 371, 372, 378, 389, 390 Standup monolayer adsorption chromatography (SMAC)������������������������������������������������������� 190, 192 Stokes shift�����������������������������������������������������������������������363 Streptavidin������������������ 95, 101, 103, 105, 107, 115, 123, 126, 130–136, 140, 141, 177, 224, 225, 227, 228, 230–235, 259, 269, 292, 294, 300, 305, 308, 309, 317, 318 Streptavidin-coupled magnetic beads���������������������� 123, 126, 130–136, 141 Surface plasmon resonance (SPR)������������������������������������111 Synthetic antibody library���������������������15, 93, 103, 366, 371 Systematic evolution of ligands by exponential enrichment (SELEX)���������������������� 253–268, 270, 271, 274, 275, 277, 278, 286, 287, 291, 292, 298, 304 T T cell receptors (TCRs)���������������������������46, 48, 55, 197–211 Therapeutics���������������������������������������5–11, 15, 67, 121, 145, 146, 175, 198, 223, 225–229, 231–235 Thermal denaturation��������������������������������������������� 47, 48, 62 Thiomabs�������������������������������������������������������������������������146 Tissue imaging���������������������������������������������������������399–413 TNF-alpha�����������������������������������������������������������������������246 Tobacco Etch Virus (TEV) protease������������������������� 95, 100, 101, 105, 113, 115, 215–218, 220 Trastuzumab (Herceptin)������������������������� 5, 9, 145–150, 162 Trifluoromethanesulphonic acid (TFMS)������������������������199 Trinucleotide-directed mutagenesis (TRIM)���������������������������������������������������� 16, 68, 345 Trypsin digestion���������������������������������������������� 176, 179–181 U Ultra performance liquid chromatography (UPLC)������������������������������������������������ 199, 201, 202 V van der Waals interactions������������������������������������������������326 Vancomycin������������������������������������������������������ 389, 391–397 VCSM13 helper phage�������������������������������������������������20, 24 Virus-like particles (VLPs)������������������������������������� 292, 294, 295, 297–301 W Western blot (WB)������������������������������������� 10, 94, 224–228, 231–233, 235 Whole cell panning (WCP)����������������������������� 67–76, 78–90 X xMAP technology������������������������������������304, 305, 307, 309, 310, 312, 314–316, 319 Y Yeast surface display�����������������������������������������������������45–63 ... (ed.), Synthetic Antibodies: Methods and Protocols, Methods in Molecular Biology, vol 1575, DOI 10.1007/978-1-4939-6857-2_1, © Springer Science+Business Media LLC 2017 Xiaoying Zhang and? ?Thirumalai Diraviyam... Antibodies: Methods and Protocols, Methods in Molecular Biology, vol 1575, DOI 10.1007/978-1-4939-6857-2_2, © Springer Science+Business Media LLC 2017 15 16 Xuelian Bai and? ?Hyunbo Shim chain framework... family involved in autoimmunity and inflammation) DigA16 (H86N) anticalin functions as a digoxin antidote when administrated intravenously in rats, dramatically decreasing the free digoxin concentration

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Từ khóa liên quan

Mục lục

  • Dedication

  • Preface

  • Contents

  • Contributors

  • Part I: Amino Acid-Based Synthetic Antibodies

    • Chapter 1: Antibody Mimetics, Peptides, and Peptidomimetics

      • 1 Introduction

      • 2 Steps Involved in Constructing Antibody Mimetics

      • 3 Antibody Mimetics as Therapeutic Agents

      • 4 Applications of Antibody Mimetics Diagnosis and Imaging

      • 5 Future Prospects for Antibody Mimetics

      • References

      • Chapter 2: Construction of a scFv Library with Synthetic, Non-­combinatorial CDR Diversity

        • 1 Introduction

        • 2 Materials

        • 3 Methods

          • 3.1 Amplification of Oligonucleotides by Polymerase Chain Reaction

          • 3.2 Construction of Single-CDR scFv Libraries

          • 3.3 Proofreading of Synthetic CDRs

          • 3.4 Construction of the Final scFv Library

          • 4 Notes

          • References

          • Chapter 3: Enzymatic Assembly for scFv Library Construction

            • 1 Introduction

            • 2 Materials

              • 2.1 Immunization

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